MATERIAL AND METHODS Seed collection and storage

Vol.53, n. 6: pp.1299-1308, November-December 2010

ISSN 1516-8913 Printed in Brazil BRAZILIAN ARCHIVES OF

BIOLOGY AND TECHNOLOGY

A N I N T E R N A T I O N A L J O U R N A L

Seed Germination of

Chresta sphaerocephala DC. and

Lessingianthus bardanoides (Less.) H. Rob. (Asteraceae)

from Cerrado

Graziela Cury

, Ana Dionísia da Luz Coelho Novembre

and Beatriz Appezzato da Glória

1

Departamento de Ciências Biológicas; Escola Superior de Agricultura ‘Luiz de Queiroz’; Universidade de São Paulo; C.P. 09; 13418-900; Piracicaba - SP - Brasil. 2Departamento de Produção Vegetal; Escola Superior de Agricultura ‘Luiz de Queiroz’; Universidade de São Paulo; C.P. 09; 13418-900; Piracicaba - SP - Brasil

ABSTRACT

This work aimed to study the effects of different temperature conditions on the germination of L. bardanoides and

C. sphaerocephala seeds, compare the germination rates of these two species and estimate the occurrence of embryoless seeds. The results indicated that the best temperatures for the germination of L. bardanoides seeds were 20, 25 and 20-30ºC and for C. sphaerocephala, 20-35ºC and 40.32 µmol m-2s-1 irradiance; L. bardanoides had a higher germination rate since the number of seeds with embryos higher compared with C. sphaerocephala.

Key words: Gemmiferous root; savanna, vegetative propagation; xylopodium

INTRODUCTION

The Cerrado biome comprises a 1.5 million km2

area within Brazil, with a diversified vegetation

ranging from campos limpos (grassland) open

country forms to cerradão (dry forests), relatively

dense forest formations. Between the two extremes, intermediate forms are found, in which

campos sujos (shrubland), campos cerrados

(brushland) or Cerrado sensu stricto (Coutinho,

2002) may be observed. In the São Paulo state territory, Cerrado occurs especially in the

mid-northern region (Toledo Filho et al., 1984), and

small cerradão spots may occur in the

mid-western region. The eastern state comprises more

open physiognomies such as campos cerrados and

campos sujos, especially preserved in the São

Carlos region (Durigan et al., 2004). The destruction in the Cerrado today is intense and,

according to Almeida et al. (2005), the flora of this biome is not fully known yet, in that in each survey new species are added to this list of biome’s angiosperm. The family Asteraceae is an important part of its flora in the herbaceous and subshrubby layers. This family includes the plants with diverse habits such as weeds, subshrubs, creepers or exceptionally trees, with most genera constituted by small-sized plants, several of them may be used as food, in pharmaceutical products, in beverage preparation, as rubber producers, or insecticides (Joly, 1998).

In the state of São Paulo, Almeida et al. (2005) performed a survey on Asteraceae species found in

eight localities of Cerrado sensu stricto and

fundamental. Among the different Asteraceae tribes listed in this study, Vernonieae was the one with the highest number of species; Appezzato-da-Glória et al. (2008) reported the presence of different thickened underground organs in two

frequent species in the state of São Paulo Cerrado

inside this tribe. In Lessingianthus bardanoides

(Less.) H. Rob., the thickened underground organ

is represented by a xylopodium and in Chresta

sphaerocephala DC., by a diffused underground

system of radicular structure that produces aerial stems (Appezzato-da-Glória et al., 2008). This underground system is an important vegetative

reproduction device in the Cerrado flora. As to the

xylopodium, according to Appezzato-da-Glória et al. (2008), it is characterized by woody consistency, self-grafting among stem branches, and budding ability. The formation may occur due to environmental influences, or may be genetically determined, and the anatomical structure may have a root, mixed or stem nature. Furthermore, the xylopodium cannot propagate vegetatively and the

only means of the reproduction for L. bardanoides

is through sexual propagation, whose efficiency not only assures the maintenance of the species but also places it as one of the most recurrent in the

São Paulo state Cerrado.

The reproductive efficiency of a species can be estimated through seed germination. There are

many studies involving Cerrado Asteraceae

germination (Achutti, 1978; Ruggiero and Zaidan, 1997; Sassaki et al., 1999; Gomes and Fernandes, 2002; Souza et al., 2003; Rosal, 2004; Velten and Garcia, 2005; Bertasso-Borges and Coleman, 2005). Neverthless, only Sassaki et al. (1999) and Velten and Garcia (2005) related the germination ratio to vegetative production.

A common phenomenon in the family Asteraceae and directly related to germination rate is the significant amount of seed production, although many of them are embryoless (Sassaki et al., 1999), which has been confirmed by others (Achutti, 1978; Sajo and Menezes, 1986; Maluf and Wizentier, 1998; Sassaki et al., 1999; Bertasso-Borges and Coleman, 2005; Velten and Garcia, 2005; Tonetti et al., 2006); this although common, has not been fully elucidated yet, and since it affects the seed germination rates, it must always be considered in germination trials of the family Asteraceae.

In order to enhance the knowledge on the herbaceous-subshrubby flora of the family Asteraceae in the São Paulo state Cerrado, thus

contributing for its preservation, the aims of this work were: (i) to study the effects of different

temperature conditions on the germination of L.

bardanoides and C. sphaerocephala seeds; (ii) to

compare the germination rates of both the species, and (iii) to estimate the occurrence of embryoless seeds for each species.

MATERIAL AND METHODS

Seed collection and storage

According to Marzinek et al. (2008) in Asteraceae, the fruit type is cypsela. Here, this term was considered as synonymous of seed. The seeds of

Lessingianthus bardanoides (Less.) H. Rob. (Fig.

1 a, b) and Chresta sphaerocephala DC. (Fig. 1 c,

d) were collected from the natural Cerrado area

populations in the state of São Paulo, Brazil, at

Estação Ecológica de Itirapina (22°13'S and

47°54'W). The time of collection varied between

the two species (June for L. bardanoides and

November for C. sphaerocephala).

The criterium of collecting was capitula in which the seeds easily removed by hand were chosen and soon after the collection, they were left to dry at room temperature (18-22ºC) for three days and then transferred to a refrigerated chamber (20ºC) with 45-50% relative humidity. The storage period varied as the experiments were set up at different

times. For the L. bardanoides experiment, the

seeds were stored for 17 days and for the C.

sphaerocephala the seed storage lasted 62 days.

Experimental set-up, seed germination and seedling development monitoring

To set up the experiments, the capitula were removed from the refrigerated chamber, the seeds were removed by hand, homogenized in a tray and randomly selected when placed for germination. Withered and preyed seeds were rejected and it was not possible to differentiate those with embryo-containing seeds from those without through aspect or by pressure.

For the L. bardanoides experiment, 25 seeds were

20–35ºC with eight-hour light in the top temperature. In all the germination chambers, the light was supplied by fluorescent light bulbs with

an irradiance of 40.32 µmol m-2_s-1_{. The C.}

sphaerocephala germination experiment was set

up following the model of the experiment of L.

bardanoides, although with 10 replications per

treatment, with 250 seeds for each temperature. The germination process was monitored weekly, from three to four days and this periodicity was defined by the low number of germinated seeds daily. When necessary, the filter paper was moistened to ensure the seeds have enough water supply throughout the test. This process was

followed during 80 days for L. bardanoides and

115 days for C. sphaerocephala. In each

observation, the emission of the primary root (Fig. 2 a) was first recorded and then the rise of the other seedling structures. For the germination, the seedlings having two cotyledons, hypocotyl and primary root were considered as normal ones. With the results of the germination tests, the germination speed was calculated by the germination speed index (GSI), according to the formula proposed by Maguire (1962) and the mean germination time (t), according to Labouriau (1983).

Analysis of nongerminated seeds

In both the experiments, following the last evaluation, all the seeds were kept under the same conditions for an additional 30-day period to verify whether there would be germinating seeds or not. At the end of this period, the experiments were stopped and all the seeds, which did not germinate, were sectioned longitudinally to verify the presence or absence of the embryo. The seeds in which a normal-appearing embryo was observed were submitted to tetrazolium test (Brasil, 1992; Velten and Garcia, 2005) to determine their viability. In this test, immediately following the longitudinal section, the seeds were placed into a small plastic cup containing 1% tetrazolium. This container was covered with aluminum sheet to prevent the evaporation of the solution and kept at 30ºC for 24 hours. The seeds with red-colored embryo were considered viable.

Statistical analysis

For the statistical analysis a completely randomized design was used and was performed by using the Sistema de Análise Estatística para

Microcomputadores (SANEST) (Zonta &

Machado, 1984). For the variance analysis, the data of germination percentage were transformed

in arc sen √_{x/100 and the Tukey test was followed}

at 5 and 1% probability for multiple comparison of means to detect the differences among the temperature treatments for each species.

RESULTS

Germination tests, analysis of embryo presence and tetrazolium test

The results of the germination tests are shown in Table 1. After performing the longitudinal section of all the seeds that failed to germinate, except for the ones killed by the fungus attack, the number of seeds without embryo, malformed embryo and nongerminated viable seeds for each temperature,

are shown in Table 2. Out of 750 L. bardanoides

seeds placed for germination, 124 seeds

germinated, and out of 1,500 C. sphaerocephala

seeds, 44 germinated. In L. bardanoides, 58% did

not have an embryo, while in C. sphaerocephala,

this was 85%. In addition to the absence of embryo and occurrence of dead seeds, another cause for the germination not to occur was the presence of a malformed embryo (Table 2). In L. bardanoides, three nongerminated seeds had a

normal looking embryo and when submitted to the tetrazolium test, revealed a red color (Fig. 2 d), thus characterizing the seeds as alive, probably viable.

Morphology of Lessingianthus bardanoides

seedlings

L. bardanoides seeds germinated normal (Fig. 2 c)

Table 1 - Total number of seeds (n), temperature, germination rate, calculation of the germination speed index (GSI) and mean germination time (t) for different temperatures and 40.32 µmol m-2_s-1 _{irradiance. Same letters in the same}

columns do not differ significantly at 5 and 1% probability.

Species n Temperature (ºC) Germination (%) GSI t (days)

125 20 27.2 (a) 0.138 (a) 46 (a)

125 25 30.4 (a) 0.214 (a) 36 (a)

L. bardanoides 125 30 11.2 (b) 0.068 (b) 39 (a)

125 15-35 7.20 (b) 0.019 (b) 78 (b)

125 20-30 18.4 (a.b) 0.094 (a.b) 43 (a)

125 20-35 4.80 (b) 0.018 (b) 51 (a)

Total 750

250 20 4.8 (a) 0.066 (a) 19 (a)

250 25 2.0 (b) 0.029 (b) 20 (a)

C. sphaerocephala 250 30 2.4 (b) 0.042 (a.b) 14 (a)

250 15-35 2.0 (b) 0.039 (a.b) 41 (b)

250 20-30 2.4 (b) 0.042 (a.b) 14 (a)

250 20-35 4.0 (a) 0.060 (a) 16 (a)

Total 1,500

Table 2 - Number of seeds with embryo, no embryo, dead seeds, malformed embryo, nongerminated viable seeds and germinated seeds ignoring the dead seeds, seeds with malformed embryos and embryoless for different temperatures and 40.32 µmol m-2_s-1 _irradiance.

Species Temperature (ºC)

With embryo

No embryo

Dead seed

Malformed embryo

Viable (alive)

Germinated (no) (%)

20 34 84 5 2 0 34 100

25 38 66 21 0 0 38 100

L. bardanoides 30 14 71 40 0 0 14 100

15-35 11 91 20 3 2 9 82

20-30 23 62 38 2 0 23 100

20-35 7 62 56 0 1 6 86

Total 127 436 180 7 3 124 16.5

20 12 203 33 2 0 12 100

25 5 223 21 1 0 5 100

C.sphaerocephala 30 6 216 24 4 0 6 100

15-35 5 208 36 1 0 5 100

20-30 6 214 29 1 0 6 100

20-35 10 221 19 0 0 10 100

Total 44 1,285 162 9 0 44 3

Table 3 - Morphological differences of L. bardanoides seedlings.

Morphology N seedlings Cotyledons Hypocotyl Primary root Adventitious root (n) (%)

abnormal 13 10.5 2 swollen degenerated hypocotyl-derived

DISCUSSION

L. bardanoides seeds had the highest germination

rates and the highest GSI values at 20, 25, and

20-30ºC. For C. sphaerocephala, the best germination

rates occurred at 20 and 20-35ºC and the highest GSI values were obtained at all the temperatures, except for 25ºC. Comparing these results with those of other studies (Ruggiero and Zaidan, 1997; Ferreira et al., 2001; Gomes and Fernandes, 2002; Velten and Garcia, 2005; Garcia et al., 2006; Tonetti et al., 2006), in which embryoless-seeds were not previously separated, within the tribe Vernonieae, the various species were observed to show distinct temperature ranges for the germination, and that variation also occurred among the germination rates in the family Asteraceae.

In relationship with the mean germination time, although there was a significant difference between the species, for both at 15-35ºC, the germination was slowest and no difference was observed at temperatures. The mean time evaluates the germination process and, as proposed by Ferreira et al. (2001), the species can be classified as rapid-germination (t < 5 days), intermediate germination (5 < t < 10 days), and slow germination (t > 10 days). According to such

classification, both L. bardanoides and C.

sphaerocephala was slow germination species.

Other species of the family Asteraceae which also

have slow germination are Trixis praestans (Vell.)

Cabrera, Senecio heterotrichius DC., S. oxyphyllus

DC. and S. selloi DC, while other species of the

tribe Vernonieae such as Elephantopus mollis H.B.

and K., Vernonia nudiflora (Less) (Ferreira et al.,

2001), Eremanthus elaeagnus (Mart. ex DC.), E.

glomeratus Less. and E. incanus (Less) Less

(Velten and Garcia, 2005) have rapid germination. Regarding the total germination of the two species,

the germination rate of L. bardanoides (16.5%)

was 5.5 times higher than that of C.

sphaerocephala (3%). This seemed plausible when

these germination rates were associated with the types of underground systems of each species. The

underground organ of L. bardanoides was made

up of one xylopodium which did not have vegetative propagation capacity, therefore, the sexual reproduction would be ensured by the

higher seed germination rate. C. sphaerocephala,

which has a low germination rate it can be propagated vegetatively through its diffused underground system of radicular structure

(Appezzato-da-Glória et al., 2008). Sprouting from root buds could represent an effective process of spatial reoccupation and is different from the sucker shoots, because these have the potential to emerge at variable distances from the main trunk, but develop their own adventitious root system, and become independent (Rodrigues et al., 2004; Hayashi and Appezzato-da-Glória, 2009).

In germination trials, Sassaki et al. (1999)

achieved a germination rate of 11% for Vernonia

herbacea (Vell) Rusby, lower than the value of

16.5% achieved in this work for L. bardanoides.

According to Hayashi and Appezzato-da-Glória

(2005), V. herbacea is one of the Cerrado species

whose underground system is a rhizophore with vegetative propagation capacity. Velten and Garcia (2005) also found low values in the

germination tests for Eremanthus elaeagnus (Mart.

Ex DC.) and attributed this to the occurrence of asexual reproduction in the species through the underground system. Moreover, in the clonal species, the production of viable seeds tends to be always low (Bell et al., 1993), which could explain

the high number of embryoless seeds in C.

sphaerocephala (85%) in relation to L.

bardanoides (58%) in this study. Sajo and

Menezes (1986) reported that in three species of

Vernonia Screb. most seeds in each capitulum did

not contain embryo. This was due to the

rhizophores that constituted vegetative

propagation units of these plants by developing new aerial branches from the parts close to the soil

surface. In Vernonia herbacea (Vell.) Rusby,

Sassaki et al. (1999) reported 85% embryoless seeds. Velten and Garcia (2005) observed 50 to

90% seeds as embryoless in Eremanthus species.

Overall, as previously stated, in Asteraceae species a higher number of embryoless seeds occurred, thus affecting the germination rate negatively. Table 2 showed a large number of embryoless seeds. At the germination trial, ignoring the dead seeds, the seeds with malformed embryos and the embryoless seeds, the germination rate was high and almost equivalent to the total number of the

seeds. Table 2 showed that for C. sphaerocephala

at all the temperatures, the germination rate was

100%, and for L. bardanoides only at two

temperatures, the germination rate was not 100% due to occurrence of nongerminated viable seeds. Achutti (1978) found only six embryo-bearing seeds (0.25%), out of 2,300 analyzed in

Piptocarpha rotundifolia (Less.) Baker.

3,632 seeds of Eupatorium laevigatum Lam.,

28.2% had an embryo. In Eremanthus

erythropappus (DC.), Tonetti et al. (2006) found

approximately 15% embryo-bearing seeds.

Many authors have discussed about the causes leading to low production of seeds with embryo in the family Asteraceae. Recently Marzinek (2008) suggested that one of the possible causes of the phenomenon was related to the abortion of ovules and seeds due to scarce maternal resources, since many flowers occurred in one capitulum. According to Lloyd (1980), however, the canceling of the maternal investment can not be considered simply as a loss of reproductive potential, instead it represented an adaptational response to the boundaries imposed by the available resources. Nevertheless, a possible cause of abortion in Asteraceae seeds was apomixis, a quite common occurrence in the family (Werpachowski et al., 2004). In capitula with cross-pollination, the development of abnormal ovules involves the proliferation of the endothelium, a failure in the formation of the embryo and failure in the endosperm development, which according to Cichan and Palser (1982) can cause the abortion of seeds in the early

development stages in Cichorium intybus L.

Self-incompatibility is frequent in family Asteraceae (Richards, 1986). Velten and Garcia (2005) suggested that this can be one of the possible causes of the reduced production of viable

seeds in Eremanthus. Ferrer and Good-Avila

(2007) reported that 63% of 572 Asteraceae species were with self-incompatibility.

As to the ecologic aspects, many Asteraceae species host insect larvae which feed on their seeds (Klinkhamer et al., 1988), and according to Fenner et al. (2002), these seeds are more available while they are attached to the capitulum and represent a source of proteins and minerals to be exploited. For Louda and Potvin (1995), in addition to the direct consumption to developing flowers and seeds in Asteraceae, which can reduce the seed production and result in lesser number of seedlings and adult individuals, the insects feed on the inflorescence in the early development stages, causing injuries to the phloem and disrupting the nutrient supply to future seeds, thus decreasing the number of viable seeds per capitulum. Louda and Potvin (1995) also reported that the capitula of

Cirsium canescens Nutt. located in the upper part,

matured before those located in the lower portion and recorded that when the herbivory interference

was experimentally reduced, a higher number of viable seeds was achieved, in view of their higher contribution from the lower part of the plant. Considering these results, two considerations were established: (1) this type of herbivory could decrease the number of viable seeds in a plant; (2) the difference between the maturation time of inflorescences within the same individual could affect significantly the seed collection, thus increasing the proportion of embryoless seeds.

The results obtained in the germination tests of L.

bardanoides indicated the presence of three seeds

that, although being viable following the tetrazolium test, failed to germinate. Among the possible causes that prevented these seeds from germinating, dormancy could be one of them. However, dormancy is inherent to the species and according to Rizzini (1997), it would achieve the germination thoroughly, which does not justify the absence of embryonal development in only three seeds. There were delays in the germination which caused fungal infestation. The presence of these microorganisms could have interfered with the germination process and prevented the three seeds from germinating.

The germination trials of L. bardanoides showed

the development of normal and abnormal seedlings. According to Brasil (1992), only the seedlings with normal development could be considered. However, the seedlings with abnormal development, according to the work mentioned above, had morphological characters such as swollen hypocotyl and emission of adventitious roots and primary root degeneration, quite similar to the xylopodium of that species found in the field. The morphological characters shown by these seedlings gave rise to a cauline xylopodium and the primary root is degenerated. Rizzini and Heringer (1961) reported that the xylopodium in

Mimosa multipinna Benth. was formed through the

thickening of the hypocotyl and the upper primary root. Other authors found through the anatomical studies (Paviani, 1977; Appezzato-da-Glória and Estelita, 2000; Hayashi and Appezzato-da-Glória,

2007) that in Brasilia sickii G.M. Barroso

(Asteraceae), Mandevilla illustris (Vell.)

Woodson, Mandevilla velutina (Mart. ex Stadelm.)

Woodson (Apocynaceae) and Vernonia

grandiflora Less. (Asteraceae), the xylopodium

ACKNOWLEDGMENTS

We are grateful to the FAPESP (Process 00/12469-3) for the financial support and to the CAPES and CNPq for grants. We also thank Instituto de Botânica and Instituto Florestal to giving the permission to collect the plant materials, Vinícius C. Souza (USP) for identifying

the species and

Orlando Cavalari de Paula

imaging the Chresta sphaerocephala.

RESUMO

Este trabalho teve como objetivo estudar os efeitos de diferentes condições de temperatura na

germinação de sementes de L. bardanoides e C.

sphaerocephala, comparar as taxas de germinação

dessas duas espécies e estimar a ocorrência de sementes sem embrião. Os resultados indicaram que as melhores temperaturas para a germinação

das sementes de L. bardanoides foram 20, 25 e

20-30ºC e, para C. sphaerocephala, 20 e 20-35ºC e

40.32 µmol m-2_s-1 _{de irradiância; L. bardanoides}

apresentou maior taxa germinativa, já que o número de frutos contendo semente com embrião é

maior, comparado com C. sphaerocephala.

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